The principal function of mitochondria is the generation of cellular energy (ATP) by oxidative phosphorylation using proteins encoded for by both the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA) to assemble the machinery needed for mitochondrial respiration. Paradoxically, mtDNA is a macromolecular target of reactive oxygen species (ROS), which are mutagenic by-products generated during ATP production. Unlike nuclear DNA, which has a high-level of proofreading, error detection and correction that coordinately function to prevent passage of harmful DNA mutations to offspring, mtDNA mutations are not subject to the same degree of detection and correction. Indeed, past studies have shown that mtDNA sustains a high mutational burden that progressively increases in cells with age. This paradigm raises a fundamentally critical question when one considers that mitochondrial passage from one generation to the next is uniparental through the female germline (egg): how are detrimental mtDNA mutations that accumulate in maternal germ cells over time prevented from being passed to the next generation, the generation after that, and so on? Two distinct, but likely interrelated, processes have been offered to explain this phenomenon ? the mtDNA bottleneck and germline-purifying selection. However, large gaps in knowledge still exist regarding both. For example, several different mechanisms have been proposed for how the mtDNA bottleneck works ? none of which have been proven, and its position in germline development is debated. Likewise, it is not known where germline-purifying selection takes place relative to the bottleneck or even if it depends on the bottleneck. We recently developed an innovative technology combining PACBIO third-generation sequencing with unique molecular identifiers, which enables high-fidelity mutational analysis of entire individual mtDNA molecules. With this in hand, we are now uniquely positioned to map how transgenerational passage of high-quality mtDNA molecules is accomplished. To do this, we propose the following Specific Aims (SAs). SA1: Define the structure and dynamics of the mtDNA bottleneck. We will build detailed phylogenetic trees of mtDNA molecules within individual germline cells, and compare these trees to those simulated from proposed bottleneck models. SA2: Determine the timing and mechanisms of germline-purifying selection. We will compare ?synonymity? of mutations from phylogenetic tree branches to identify when purifying selection occurs, and how it interfaces with the bottleneck. SA3: Determine if individual mtDNA molecules carrying no/low versus high mutational burdens are preferentially allocated during early (preimplantation) embryogenesis. We will evaluate mtDNA genealogies in individual blastomeres of preimplantation embryos at the 2-, 4- and 8-cell stages, as well as in the inner cell mass (embryo proper) and trophectoderm (extraembryonic) of blastocyst-stage preimplantation embryos, to determine if there exists evidence of differential allocation of mtDNA mutations.